Cell penetrating peptides (CPPs) can cross cell membranes in a receptor-independent manner. Two main routes for their cellular uptake have been proposed: endocytosis and direct translocation through the cell membrane. The ability of a peptide to enter cells through direct translocation can be assessed by evaluating the amount of peptide crossing the membrane of liposomes. Most methods reported so far rely on the use of fluorescent probes, which when attached to a CPP, often alter its physical/chemical properties. Herein, a MALDI-TOF MS-based method is described to quantify the amount of CPP taken up into lipid vesicles and to distinguish it from the amount that is bound or inserted in the membrane. As a comparison, visualization of the uptake of the same, but fluorophore-labeled, peptides into giant vesicles and cells by fluorescence microscopy is also reported. We show that membrane charge density is an important factor for direct translocation. We also show that fluorophore-labeled peptides have a different translocation behavior and that they are more toxic to cells. Alternative methods to fluorescence, such as the one reported herein should be favored when investigating the uptake mechanism of CPPs, as fluorescent dyes can alter short peptides physical/chemical properties and their internalization capacities.